JP2008509407A - Use of transthyretin as a biomarker for colorectal adenoma and / or carcinoma; detection method and test system - Google Patents

Abstract

The invention is directed to a method for detecting colorectal adenoma and/or colorectal carcinoma comprising the steps: a) providing an isolated sample material which has been taken from an individual, b) determining the level of transthyretin in said isolated sample material, and c)comparing the determined level of transthyretin with a reference value. The invention is further directed to a method for discriminating between colorectal adenoma and colorectal carcinoma as well as to a method for monitoring the course of colorectal adenoma and/or colorectal carcinoma and/or the treatment of colorectal adenoma and/or colorectal carcinoma. Moreover, the invention is directed to a test system and an array for use in these methods. Furthermore, the invention is directed to the use of transthyretin as a biomarker for a detection of colorectal adenoma and/or colorectal carcinoma in an individual.

Description

  The present invention relates to the field of detection of colorectal adenoma and / or colorectal carcinoma.

  Colorectal carcinoma is the third most frequently diagnosed carcinoma in the West and is the second leading cause of cancer death. In 1996, an estimated 133500 colorectal carcinoma cases were newly diagnosed in the United States, and approximately 54,900 people died from the disease. The incidence of colorectal carcinoma is increasing, while the mortality rate of colorectal carcinoma is decreasing. The incidence of colorectal carcinoma has increased with age since about 40 years, and this rate is higher in males than in females (60.4 males versus 40.9 females per 100,000 per year).

  In most patients, the development of colorectal carcinoma occurs after a multi-stage progression from precancerous adenoma to invasive malignancy with propensity to metastasis. Decreased morbidity and mortality of colorectal carcinoma has been demonstrated to be achieved by the detection and treatment of early colorectal carcinoma and the identification and removal of colorectal adenomatous polyps that are precursors of colorectal carcinoma .

  Colorectal screening studies have shown that accurate detection of its precursors, namely adenomatous polyps and / or squamous neoplastic areas, can be achieved. Several trials are available as options for colorectal carcinoma screening. Screening tests include fecal occult blood test (FOBT), flexible sigmoidoscopy, combined use of FOBT and flexible sigmoidoscopy, and colonoscopy. Various screening tests differ from each other in terms of performance, effectiveness, possible screening frequency, test complexity, price, and patient acceptance.

  At present, screening by the fecal occult blood test is considered to be the optimal screening strategy in terms of cost effectiveness. Fecal occult blood can be detected by chemical reagents such as guaiac, through heme porphyrin, or by immunological methods. The guaiac slide test Hemocult (II) available from SmithKline Diagnostics is the most widely used.

  Various technical factors affect its clinical performance. Hemocult has a sensitivity of about 50% for colorectal carcinoma and a specificity of about 98%, but a sensitivity of about 10% for polyps (Simon JB. (1998) Gastroenterologist, 6: 66-78. Review). . Another significant drawback of occult blood screening is the low accuracy of predictions, and it has been found that only 10% of positive reactions are due to colorectal carcinomas (Simon JB. (1998) Gastroenterologist, 6: 66-78.Review; Mandel JS et al. (1999) J. Natl. Cancer Inst., 91: 434-437; Hardcastle JD et al. (1996) Lancet, 348: 1472-1477; Kronborg O et al. 348: 1467-71; Winewer SJ et al. (1997) Gastroenterology, 112: 594-642; Fletcher RH (1998) N. Engl. J. Med., 338: 1153- 154).

  In addition, fecal occult blood tests only provide results after the disease has progressed to a specific stage. It would be desirable to have a test system that allows detection of colorectal adenoma and / or colorectal carcinoma at an earlier time point.

  More recently developed immunological tests are generally sensitive but have poor properties and still have serious problems. Other methods such as genetic testing of stool samples for the KRAS oncogene and p53 protein are still ineffective (Calistri D et al. (2003) Clin. Gastroenterol. Hepatol., 1: 377-383; Schoen RE (2002) ) Nat.Rev.Cancer, 2: 65-70).

  Endoscopy (Kavanag AM (1998) Cancer Causes Control) using either a flexible sigmoidoscope or colonoscope (Lieberman DA (1997) Gastroenterol. Clin. N. Am., 26: 71-83). 9: 455-462) is the most definitive detection means, but has limitations. False negatives for squamous tumor lesions have been observed and the rate remains high (Kudo S (1997) Gastrointest. Endosc. Clin. N. Am., 7: 87-98). Colonoscopy allows examination of the colon with a low false negative rate for polypoid lesions with a diameter of at least 10 mm. For this reason, the interval between the negative determination and the re-examination can be a relatively long period of up to 10 years, or up to 5 years after polypectomy.

  However, if such a re-examination after colonoscopy is recommended, the patient is unlikely to accept. Furthermore, colonoscopy is expensive and cumbersome. Due to the high cost of generalized examinations and the limited awareness of colonoscopy by people, the application of this examination method is limited.

  Collected isolated tissue samples can be tested by various methods for colorectal carcinoma and its precursor, colorectal adenoma. DE 197 11 111A discloses a method using in vitro determination of intraepithelial colonic bacteria, components and their reaction products. Another method using HERG gene expression in tissue samples is disclosed in DE 102 24 534.

  It is an object of the present invention to provide a means that allows early detection of colon adenoma and / or colon carcinoma.

  It is a further object to provide a biomarker that can be used in the detection of colorectal adenoma and / or carcinoma.

  Another object of the present invention is to provide a test system for detecting colorectal adenomas or carcinomas that is cost effective and can be widely used.

  Furthermore, the test system should be simple to operate and more convenient for individuals examining colorectal adenomas and / or carcinomas.

  The object underlying the present invention is solved by the use of transthyretin as a biomarker for the detection of colorectal adenoma and / or colorectal carcinoma in an individual. Detection can be performed both in vivo and in vitro. According to a preferred embodiment, this detection is performed in vitro.

The object of the present invention is further a method for detecting colorectal adenoma and / or colorectal carcinoma comprising:
a) Prepare an isolated test material collected from the individual,
b) quantifying the level of transthyretin in the isolated test material,
c) comparing the quantified level of transthyretin to a reference value;
Solved by a method comprising steps.

The object of the present invention is further a method for distinguishing between colorectal adenoma and colorectal carcinoma comprising:
a) Prepare an isolated test material collected from the individual,
b) quantifying the level of transthyretin in the isolated test material,
c) comparing the quantified level of transthyretin to a reference value;
Solved by a method comprising steps.

The object of the present invention is a method for monitoring the course of colorectal adenoma and / or colorectal carcinoma and / or the treatment of colorectal adenoma and / or colorectal carcinoma:
a) Prepare an isolated test material collected from the individual,
b) quantifying the level of transthyretin in the isolated test material,
c) comparing the quantified level of transthyretin to a reference value;
It is also solved by a method including steps.

The object of the present invention is also a test system for detecting colorectal adenoma and / or colorectal carcinoma in an individual comprising:
a) an antibody or receptor that binds to an epitope of transthyretin;
b) a solid support that supports the antibody or receptor;
c) a reagent for detecting the binding of the epitope of the transthyretin to the antibody or receptor;
It is also solved by providing a test system including:

The object of the present invention is also an array comprising detection molecules for detecting colorectal adenoma and / or colorectal carcinoma in an individual, comprising:
a) a nucleic acid probe immobilized on a solid support for binding to and detecting transthyretin-encoding mRNA, or b) a solid support for binding to and detecting an epitope of transthyretin An immobilized antibody, or c) a receptor immobilized on a solid support for binding to and detecting an epitope of transthyretin,
This is preferably solved by providing an array in which each different amount of detection molecule is immobilized on a solid support to increase the accuracy of quantification.

  Preferred embodiments are specified in the dependent claims.

  According to the present invention, the term “biomarker” is meant to indicate a protein or protein fragment that is an indicator for the development of colorectal adenoma and / or colorectal carcinoma. This means that “biomarkers” are used as detection agents or detection molecules.

  The term “individual” or “individual” has the meaning to indicate a mammal. Preferably, the mammal is a human such as a patient.

  The term “healthy individual” or “healthy individual (s)” is meant to indicate an individual (s) not suffering from colorectal adenoma and / or colorectal carcinoma. That is, the term “healthy individual (s)” is used only with respect to the pathological state of colorectal adenoma and / or colorectal carcinoma and suffers from diseases other than colorectal adenoma and / or colorectal carcinoma. It does not exclude individuals that are present.

  As used herein, the term “transthyretin” also includes a truncated transthyretin, a fragment of transthyretin, a mutant transthyretin, or a modified transthyretin. The modification of “transthyretin” may be due to enzymatic or chemical modification. Furthermore, the term “transthyretin” is also used to indicate a monomeric or multi-form of transthyretin. For example, the term “transthyretin” specifically includes monomeric protein chains that are usually part of the homotetrameric protein transthyretin.

  The term “epitope” is intended to indicate any antibody, antibody fragment, protein or peptide structure, or any structural element of transthyretin that allows specific binding of a receptor.

  The process of isolating the test material is not part of the present invention. That is, the method of the present invention is performed on a test material such as a body fluid that has already been isolated. For example, it is possible to store the specimen material to be tested in a freezer and thaw each specimen material before carrying out the method of the invention at an appropriate time.

  Surprisingly, it has been found by the inventors that the protein transthyretin can be used as a biomarker for the detection of colorectal adenoma and / or carcinoma.

  Transthyretin has also been named prealbumin. Transthyretin is a tetrameric protein with a molecular weight of approximately 54 kDa, synthesized primarily in the liver. Transthyretin is composed of four protein chains of about 14 kDa that can be identical. That is, transthyretin can be considered as a homotetrameric protein. It functions primarily as a binding protein for thyroxine and as a carrier protein for retinal binding protein, which in turn functions as a transport protein for vitamin A (Schussler, GC, Thyroid. (2000), 10: 372; Hamilton, JA and Benson, MD, Cell of Life Sci. (2001) 58: 1491-1521).

  Transthyretin has also been reported to form a complex with amyloid β-protein. Amyloid β-protein aggregation is associated with amyloid deposition in the brain, which is a pathological feature of Alzheimer's disease.

  Transthyretin has also been reported as an indicator of nutritional status, as serum levels of transthyretin are reduced by inappropriate protein intake and acute or chronic inflammation. In addition, transthyretin is suitable for monitoring nutritional status due to its short half-life of only 2-3 days (Pleban WE, Conn. Med. (1989) 53: 405-407; Bernstein LH). And Ingenbleek Y, Clin. Chem. Lab. Med. (2002) 40: 1344-1348).

  WO 02/059621 discloses reagents that modulate transthyretin and reagents that bind to the transthyretin gene product and thereby play a role in preventing, ameliorating or correcting obesity and related dysfunction.

  By SELDI (surface enhanced laser desorption ionization) analysis, monomeric transthyretin protein chains are generated in human plasma or serum in at least two variants with molecular weights of 13.7376 Da and 13.858 Da, respectively It has been shown.

  Different molecular weights are thought to arise due to various post-translational modifications such as notches, cysteinylation, and / or glutathioneation. Cysteinylated transthyretin has a molecular weight of about 13.871 ± 11 Da, and glutathioneated transthyretin has a molecular weight of about 14.069 ± 5 Da.

  WO 03/001182 discloses the detection and isolation of homocysteinylated transthyretin from biological fluids and its use as a diagnostic marker for homocysteineemia, hyperhomocysteinemia and related diseases. ing.

  In several studies, transthyretin has been discussed as a diagnostic marker for carcinomas. Suresh et al. (J. Clin. Pathol. (1991) 44: 573-575) found that transthyretin was a useful marker in bronchopulmonary carcinoid tumors because 75% of carcinoid tumors were positive for transthyretin. Reporting. In another study, transthyretin has been discussed as a prognostic marker in epithelial ovarian carcinoma (Mahlck et al., Gynecol. Obstet. Invest. (1994) 37: 135-140).

  The inventors have surprisingly found that the level of transthyretin in bodily fluids correlates with the development of colorectal adenoma and / or carcinoma in the individual.

  According to the present invention, transthyretin is contained in body fluids such as blood, blood plasma, blood serum, bone marrow, feces, synovial fluid, lymph fluid, cerebrospinal fluid, sputum, urine, breast milk, semen, exudates, and mixtures thereof. Can be measured.

  Preferably, the body fluid is isolated prior to performing the method of the present invention. The method of the present invention is preferably performed in vitro by a laboratory technician.

  According to a preferred embodiment of the invention, transthyretin in blood plasma or blood serum is measured. Blood serum can be easily obtained by collecting blood from an individual to be medically examined and separating the supernatant from the clotted blood.

  The level of transthyretin in the body fluid, preferably blood serum, decreases with progressive formation of colorectal adenoma. Colorectal adenomas are benign tumors that can become malignant. When developing colorectal cancer from a benign colorectal adenoma, the level of transthyretin in the body fluid, preferably blood serum, is further reduced.

  After transformation of colorectal adenoma to colorectal cancer, the pathological state of the affected individual can be further exacerbated by metastasis formation.

  The present invention provides an early stage biomarker that makes it possible to detect neoplastic diseases at an early still benign stage. Early detection allows doctors to remove colorectal adenomas in a timely manner and dramatically increase the likelihood that an individual will survive.

  Furthermore, the present invention allows the level of transthyretin in body fluids such as blood serum to be monitored over a long period of time, such as yearly.

  Long-term monitoring makes it possible to distinguish between colorectal adenoma and colorectal carcinoma. The level of transthyretin can be checked periodically, for example once or twice a year. If a decrease in the level of transthyretin is detected, this can be an indicator of colorectal adenoma. Subsequent further reductions in transthyretin levels can be indicative of transformation to malignant colorectal carcinoma.

  In addition, the course of disease and / or treatment can be monitored. For example, if the level of transthyretin further decreases after removal of colorectal adenoma, this can be an indicator of worsening of the pathological condition.

  That is, the level of transthyretin is a valuable clinical parameter for the detection and / or monitoring of colorectal adenoma and / or colorectal carcinoma. Transthyretin levels in body fluids decrease very early after the onset of colorectal adenoma. Thus, transthyretin or transthyretin levels is an important clinical parameter that allows early diagnosis and thereby early treatment of disease.

  The method of the present invention for the detection of colorectal adenoma and / or colorectal carcinoma comprises preparing an isolated test material taken from an individual and then quantifying the level of transthyretin in the isolated test material. And finally, a step of comparing the quantified level of transthyretin to a reference value.

  The reference value can be calculated as an average level of transthyretin quantified in an isolated sample of a plurality of healthy individuals. This reference value can be established as a range that is considered normal, meaning that the person is healthy. Thus, a decrease below this range can be indicative of the pathological status of colorectal adenoma and / or colorectal carcinoma. This range of reference values should be established by taking a statistically appropriate number of body fluid samples, such as serum samples of healthy individuals, to be performed in any other medical parameter range, such as blood glucose, for example. Can do.

  In another embodiment of the present invention, the reference value may be an individual reference value calculated as an average level of transthyretin quantified in a plurality of isolated samples taken from the individual over time.

  When monitoring transthyretin levels for extended periods such as over a month or over a year, it is possible to establish an average level for the individual. Transthyretin levels can be measured, for example, from the same blood serum sample that is used to measure blood glucose, and this can be used to specifically detect any decrease in individuals of transthyretin levels An individual calibration curve can be established.

In another aspect, the present invention further provides a test system for detecting colorectal adenoma and / or colorectal carcinoma in an individual. The test system is based on the specificity of any antibody or receptor that binds to an epitope of transthyretin or a fragment of transthyretin or a suitable structural element. The receptor can take any structure capable of specifically binding to transthyretin. The receptor can be, for example, an antibody fragment such as a Fab or F (ab ′) ₂ fragment, or any other protein or peptide structure capable of specifically binding to transthyretin.

  The antibody, antibody fragment or receptor is bound to a solid support, such as, for example, a plastic surface or bead to allow binding and detection of transthyretin. For example, a conventional microtiter plate can be used as the plastic surface. Detection of transthyretin binding can be accomplished, for example, by using a secondary antibody labeled with a detectable group. A detectable group is an enzyme, such as horseradish peroxidase or alkaline phosphatase, which can be detected, for example, by adding a radioisotope or a suitable substrate, for example by producing a chromogenic or fluorescent signal. sell.

  The test system can be an immunoassay such as an enzyme linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). However, any other immunological test system that uses the specificity of antibodies or antibody fragments, such as Western blotting or immunoprecipitation, can be used.

  The invention also provides an array comprising a detection molecule for detecting colorectal adenoma and / or colorectal carcinoma in an individual, wherein the detection molecule comprises transthyretin, a fragment, a mutant, a variant or a derivative thereof. A nucleic acid probe immobilized on a solid support to bind to and detect the encoded mRNA, or an antibody immobilized on the solid support to bind to and detect an epitope of transthyretin, Alternatively, an array is provided which can be receptors immobilized on a solid support to bind to and detect epitopes of transthyretin.

  The nucleic acid probe may be any of natural or synthetic oligonucleotide, cDNA, cRNA and the like.

The array can be used in the test system of the present invention. The array can be either a microarray or a macroarray. The support can be a polymeric material such as nylon or plastic, or an inorganic material such as silicon, for example a silicon wafer, or ceramic. According to a preferred embodiment, glass (SiO ₂ ) is used as the solid support material. The glass can be a glass slide or a glass chip. According to another embodiment of the invention, the glass substrate has an atomically flat surface.

  For example, an array can be composed of immobilized nucleic acid probes that can specifically bind to transthyretin mRNA present in body fluids such as serum. Another preferred embodiment consists in producing cDNA by reverse transcription of transthyretin mRNA and specifically detecting the amount of each cDNA in the array. Array technology is known to those skilled in the art. Quantification of the measured mRNA or cDNA can be performed by comparing the measured value with a standard or standard curve of a known amount of transthyretin mRNA or cDNA, respectively.

  Preferably, different amounts of detection molecules are each immobilized on a solid support to allow accurate quantification of transthyretin levels.

  According to another embodiment of the invention, the level of transthyretin is quantified by mass spectrometry.

  Mass spectrometry makes it possible to specifically detect transthyretin or its monomeric protein chain based on its molecular weight and to quantify the amount of transthyretin very easily.

  Transthyretin is usually a homotetramer containing four protein chains each having a molecular weight of about 14 kDa.

  The inventors have used mass spectrometry to detect several variants of transthyretin protein chains having a molecular weight of, inter alia, 13.776 Da, 13.884 Da or 14.103 Da.

  We have reduced levels of molecular variants of transthyretin having molecular weights of 13.776 Da and 13.884 Da, particularly in body fluids such as serum in the case of the development of colorectal adenoma and / or colorectal carcinoma I have found that.

  Any suitable ionization method known in the art in the field of mass spectrometry can be employed to ionize transthyretin molecules, fragments, mutants, variants or derivatives thereof. Examples of ionization methods include electron impact (EI), chemical ionization (CI), field ionization (FDI), electrospray ionization (ESI), laser desorption ionization (LDI), and matrix-assisted laser desorption ionization. Method (MALDI) and surface enhanced laser desorption ionization (SELDI).

  Any suitable detection method conventionally known in the field of mass spectrometry can be employed to determine the molecular weight of transthyretin, fragments, mutants, variants or derivatives thereof. Examples of the detection method include quadrupole mass spectrometry (QMS), Fourier transform mass spectrometry (FT-MS), and time-of-flight mass spectrometry (TOF-MS).

  Preferably, the mass spectrometry is surface enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS). Prior to performing SELDI-TOF-MS, the transthyretin in the isolated sample is preferably immobilized on a chip or solid support with an activated surface. The activated surface preferably comprises an immobilized anti-transthyretin antibody, such as a rabbit polyclonal anti-transthyretin antibody. Following the binding of transthyretin to the antibody, a time-of-flight analysis is performed on a SELDI-TOF mass spectrometer, which delivers an intensity signal for quantification of transthyretin levels.

  Furthermore, mass spectrometry allows the simultaneous detection of other proteins that may be relevant for the detection of colorectal adenoma and / or colorectal cancer.

  In another embodiment of the invention, the sensitivity and / or specificity of detecting colorectal adenoma and / or colorectal carcinoma is enhanced by detecting another protein in combination with transthyretin.

  Other biomarkers for colorectal adenoma and / or colorectal carcinoma that can be quantified in addition to the level of transthyretin include proteins or polypeptides having a molecular weight of 8.960 Da. The level of the 8.960 Da protein or polypeptide in the test material is a colorectal adenoma and / or colorectal carcinoma when compared to the level of the protein or polypeptide in a sample isolated from a healthy individual. Has been found to increase.

  Therefore, it is preferable to quantify the level of the protein having a molecular weight of 8960 ± 9 Da in addition to the level of transthyretin in body fluids such as serum by mass spectrometry. Mass spectrometry is an optimal method for detecting and quantifying the 8960 ± 9 Da protein level.

  In another embodiment of the invention, the sensitivity and / or specificity of detection of colorectal adenoma and / or colorectal carcinoma is CEA (carcinoembryonic antigen) and / or CA 19-9 in combination with transthyretin. Enhance by detecting. Both CEA and CA 19-9 were analyzed as responsiveness indicators and prognostic factors in advanced colorectal carcinoma.

  In a further embodiment of the invention, the sensitivity and / or specificity of detection of colorectal adenoma and / or colorectal carcinoma is CA15-3, CA-125 and / or Her-2 / neu in combination with transthyretin. Enhance by detecting. CA15-3 is an oncofetal antigen that is expressed by various carcinomas and is often measured along with other tumor markers. Both CA15-3 and CA-125 are primarily prognostic indicators for breast cancer, but are also prognostic indicators for visceral metastases. Her-2 / neu amplification in breast cancer is associated with poor prognosis, short disease-free duration, and short survival time. To date, little is known about the starting point and progress of Her-2 / neu amplification.

  The methods of the invention can be performed in combination with other diagnostic methods for the detection of colorectal adenoma and / or colorectal carcinoma to increase overall sensitivity and / or specificity.

  Preferably, the method of the invention is performed as an early detection and / or monitoring method. If the results of the method of the invention indicate the development of colorectal adenoma and / or colorectal adenoma, further examinations such as colonoscopy or fecal occult blood test (FOBT) should be performed.

  In practicing the present invention, the following polyclonal anti-transthyretin antibody: The Binding Site Ltd. PC 066 available from Birmingham, UK, and A 0002 available from DAKO, Hamburg, Germany.

  The following drawings and examples are given for illustrative purposes only. The present invention should not be construed as limited to the following examples.

  Unless otherwise stated, all methods were performed according to the analytical system manufacturer's protocol.

Serum collection and serum fraction Serum was collected from three groups of human patients and examined.

  The first group consisted of 30 patients who were surgical patients treated for non-cancerous diseases such as inguinal hernia, gallstones or diverticulitis. The first group of these individuals was taken as a group of healthy individuals, ie individuals not suffering from colorectal adenoma and / or colorectal carcinoma.

  Group 2 consisted of 29 patients, all surgical patients who had been treated for indefinite tumors and found to be benign colorectal adenoma.

  Group 3 consisted of 28 patients who were patients with colorectal carcinoma. All these 28 patients suffered from TNM stage III (tumor, nodule, metastasis stage III) colorectal carcinoma.

  Strict adherence to ethical guidelines and patient confidentiality, and all patients obtained written consent to participate in the study. All patients received equivalent preoperative preparations such as fasting time and premedication.

  Serum from each patient was fractionated by anion exchange chromatography (Serum Fractionation Kit / Q HyperD resin, Ciphergen Biosystems, Inc.) using a 96-well automated approach (Biomek2000, Ciphergen) according to the manufacturer's protocol. And reduced some of the interferences caused by the most abundant proteins. As shown in FIG. 1, six fractions containing proteins roughly separated based on the pI values of the proteins were obtained by fractionation.

  From 8 patients, Ciphergen Biosystems, Inc. All fractions were tested using a chromatographic surface ProteinChip® array. The array provides a relatively high capacity and binds a wide variety of proteins and peptides for a wide range of protein profiling and peptide mapping applications. Six serum fractions from eight patients were divided into the following three different protein arrays: strong anion array with quaternary amine function (SAX-2), immobilized metal with nitriloacetic acid surface It was applied to a capture array (IMAC) and a weak cation exchange array (CM10) with carboxylate functional groups. The two fractions (Fraction 1, Fraction 4) that gave the highest evaluable peak were selected for the profiling approach on the chip (CM10 array) that showed the lowest data variability. These two fractions were measured in all three groups using a Ciphergen ProteinChip® Array CM10.

Chiphergen ProteinChip® Array Preparation and SELDI-TOF-MS Analysis CM10 protein arrays were processed with a bioprocessor (Ciphergen Biosystems, Inc.) according to the manufacturer's protocol. The chip was equilibrated with CM10 binding buffer (Ciphergen Biosystems, Inc.) for 2 × 5 minutes and then incubated with a serum fraction that had been diluted 1:10 with CM10 binding buffer. After 45 minutes, unbound material was removed and the chip was washed 3 times with CM10 binding buffer and 2 times with water. After drying at room temperature for 10 minutes, 0.05M sinapinic acid (1.0 μl) was added 2 times and the chip was analyzed with Ciphergen Protein ChipReader (model PBSII).

  Protein ChipReader is a time-of-flight mass spectrometer. Mass values and signal intensities for the detection proteins are transferred to the software, which is supplied by Ciphergen for further analysis by the ProteinChip Data Analysis Program and Biomarker Wizard Program.

  To minimize data variability, measurements were performed within 2 days using samples from all patient groups distributed randomly on the chip. As a reference control for normalization, stored normal serum was used in parallel for all measurements.

  Protein mass spectra were generated by using 195 average laser shots at 185 laser intensity. The detector was operated with a sensitivity of 7. In order to obtain data, the detection size range was set between 2.000 and 40.000 Da. The laser was focused at 10.000 Da. Data were analyzed with ProteinChip Data Analysis Program (version 3.1, Ciphergen Biosystems) and Biomarker Wizard Program (version 3.1, Ciphergen Biosystems). Peak intensity was normalized to the total ion current.

Results In this experiment, differentially expressed between sera from non-carcinoma patients (Group 1) and sera from patients with colorectal adenoma (Group 2) and colorectal carcinoma (Group 3) A protein was identified. As shown in Table 1, the six peaks showing the mean value of the intensity of high significant difference (p ≦ 0.0000001) among healthy patients (non-cancer patients), colorectal adenoma patients and colorectal carcinoma patients are fraction 1 Could be identified. As shown in Table 2, Fraction 4 provided an additional set of potential biomarkers that were significantly different between the three groups (p ≦ 0.0000001).

Table 1:
Biomarkers selected after treatment of fraction 1 of each serum sample on Ciphergen chips. The p value was obtained in a non-parametric test from the application of Biomarker Wizard. N represents a “healthy” patient, A represents a colorectal adenoma patient, and Ca represents a transthyretin level for a colorectal carcinoma patient. StDev is the standard deviation of the measured value.

Table 2:
Biomarkers selected after processing of fraction 4 of each serum sample on Ciphergen chip. The p value was obtained in a non-parametric test from the application of Biomarker Wizard. N represents a “healthy” patient, A represents a colorectal adenoma patient, and Ca represents a transthyretin level for a colorectal carcinoma patient. StDev is the standard deviation of the measured value.

  Several proteins were tested using a Biomarker Pattern Software as a single biomarker and in combination with other markers. One protein having a molecular weight of 13.770 Da (p13.770) was found to discriminate between adenoma / carcinoma patients and healthy controls, with a sensitivity of 75% and a specificity of 90%.

  FIG. 2 shows a SELDI profile illustrating a biomarker. FIG. 3 shows the discrimination of healthy patients from adenoma / carcinoma patients in a scatter plot. The intensity of the biomarker with a molecular weight of 13770 Da (p13.370) is significantly lower in the serum of adenoma / cancer patients than in healthy patients.

  FIG. 4 shows the SELDI-TOF protein profile of the fourth serum fraction from healthy, adenoma and cancer patients. As can be seen, the intensity of the p13.370 peak is significantly reduced from healthy patients to colorectal adenoma patients to colorectal cancer patients.

  When combined with a second marker protein (molecular weight 8.960 Da, first fraction), the separation of healthy patients from adenoma / carcinoma patients was significantly improved. Thirty non-adenoma / carcinoma (healthy) patients, 20 of 29 colorectal adenoma patients and even 20 of 28 colorectal carcinoma patients were correctly isolated. This combination yielded 70% sensitivity and 100% specificity.

Identification of transthyretin A 13.770 Da protein was identified as transthyretin using immunoaffinity capture and immunoprecipitation tests.

Protein Chip Immunoaffinity Capture of Transthyretin Immunoaffinity capture was performed using Protein G Array (Ciphergen Biosystems, Inc.) according to the manufacturer's protocol. Rabbit polyclonal anti-transthyretin antibody A0002 (Dako, Hamburg, Germany) was diluted to a concentration of 0.2 μg / μl with PBS. 2 μl per spot was pipetted onto the array and subsequently incubated for 24 hours at 4 ° C. in a humid chamber. Unbound antibody was removed by washing with wash buffer (PBS containing 0.5% (volume / volume) Triton X-100). After drying, the array was inserted into a sample incubation bioprocessor (Ciphergen Biosystems, Inc.). 2 μl of serum fraction (colorectal adenoma / carcinoma patient vs. healthy patient) containing large and small amounts of 13.770 Da protein was added to the chip and incubated for 1 hour at room temperature on a horizontal shaker. After washing with PBS and HEPES buffer, the array was air dried for 10 minutes and 0.6 μl of saturated sinapic acid matrix was added to each spot. The array was measured using the following parameters. The laser intensity was set to 185 and the sensitivity was set to 7. The detection size range was set between 200 and 200.000 Da, and the laser was focused at 10000 Da.

  After binding to the antibody-coated array, four protein peaks of different molecular weights (approximately 13.761 Da, 13.914 Da, 14.102 Da and 14.292 Da) have been identified and transthyretin is a different molecular variant It was suggested that FIG. 5 shows a comparison of signal intensity between healthy patients and carcinoma patients.

  Finally, immunoprecipitation was performed. 50 μl of fraction 4 serum from different patients was incubated for 2 hours at 4 ° C. with 15 μl (6,75 μg) of polyclonal antibody against transthyretin. Negative controls were performed in the same way without antibody. 50 μl protein A / G agarose (Pierce, Rockford, IL) was added and incubated overnight at 4 ° C. After centrifugation at 3300 xg, the supernatant was removed and stored separately. The pellet was resuspended in 20 μl of buffer (9 M urea, 2% Chaps, 50 mM Tris-HCl, pH 9) to remove protein from protein A / G-agarose. Both sediment and supernatant were diluted 1:10 in CM10 binding buffer. Three proteins of different molecular weights (13.776 Da, 13.884 Da, 14.103 Da) were immunoprecipitated with anti-transthyretin antibodies. From FIG. 6, the amount of the two variants (13.776 Da and 13.884 Da) was significantly reduced in the supernatant while the amount of the variant with the highest molecular weight (14.103 Da) remained unchanged. I can see that.

  This indicates that transthyretin protein chains with molecular weights of about 13.776 Da and about 13.884 Da are important biomarkers for the detection of colorectal adenoma and / or colorectal carcinoma.

It is a schematic diagram which shows the fractionation and profiling of a serum sample. It is a figure which shows a SELDI-TOF profile. It is a figure which shows the distinction of the healthy individual from the individual of adenoma / carcinoma. It is a figure which shows the SELDI-TOF protein profile of the serum fraction from three different individuals. FIG. 5 shows SELDI immunoaffinity capture of transthyretin and its potential derivatives. It is a figure which shows the immunoprecipitation using an anti-transthyretin antibody.

Claims (28)

A method for detecting colorectal adenoma and / or colorectal carcinoma comprising:
a) Prepare an isolated test material collected from the individual,
b) quantifying the level of transthyretin in the isolated test material,
c) comparing the quantified level of transthyretin to a reference value;
A method comprising the steps. A method for distinguishing between colorectal adenoma and colorectal carcinoma comprising:
a) Prepare an isolated test material collected from the individual,
b) quantifying the level of transthyretin in the isolated test material,
c) comparing the quantified level of transthyretin to a reference value;
A method comprising the steps. A method for monitoring the course of colorectal adenoma and / or colorectal carcinoma and / or the treatment of colorectal adenoma and / or colorectal carcinoma:
a) Prepare an isolated test material collected from the individual,
b) quantifying the level of transthyretin in the isolated test material,
c) comparing the quantified level of transthyretin to a reference value;
A method comprising the steps.   The method according to any one of claims 1 to 3, wherein the level of transthyretin in the test material is decreased as compared to the test material of a healthy individual.   A first decrease in the level of transthyretin in the first test material is indicative of colorectal adenoma and in a second test material isolated from the individual at a later time than the first test material. 5. A method according to any one of claims 2 to 4, wherein a second decrease in the level of transthyretin is indicative of colorectal carcinoma, wherein the second decrease is greater than the first decrease.   The method according to any one of claims 1 to 5, wherein the reference value is calculated as an average level of transthyretin quantified in an isolated sample of a plurality of healthy individuals.   The method according to any one of claims 1 to 5, wherein the reference value is an individual reference value calculated as an average level of transthyretin quantified in a plurality of isolated samples collected from an individual over a long period of time. .   The method according to any one of claims 1 to 7, wherein the transthyretin is composed of one or four protein chains which may be the same or different from each other.   9. The method according to any one of claims 1 to 8, wherein the transthyretin is notched, modified and / or mutated.   The method according to any one of claims 1 to 9, wherein the isolated test material is a body fluid.   11. The body fluid according to claim 10, wherein the body fluid is selected from the group consisting of blood, blood plasma, serum, bone marrow, stool, synovial fluid, lymph fluid, cerebrospinal fluid, sputum, urine, breast milk, semen, exudate, and mixtures thereof. Method.   The method according to any one of the preceding claims, wherein the level of transthyretin in the test material is quantified by mass spectrometry.   The level of carcinoembryonic antigen (CEA) in the test material is further quantified, and the level of carcinoembryonic antigen (CEA) in the test material is increased by colorectal adenoma and / or colorectal carcinoma. The method of any one of 1-12.   14. The level of CA 19-9 in the test material is further quantified, and the level of CA 19-9 in the test material is increased by colorectal adenoma and / or colorectal carcinoma. 2. The method according to item 1.   The level of a protein or polypeptide having a molecular weight of 8960 ± 9 Da is further quantified, and the level of a protein or polypeptide of 8960 ± 9 Da in the test material is increased by colorectal adenoma and / or colorectal carcinoma. The method of any one of -14.   12. The method according to any one of claims 1 to 11, wherein the level of transthyretin in the test material is quantified by immunoassay, preferably by ELISA or RIA.   The level of transthyretin in a test material is quantified by quantifying the level of mRNA encoding transthyretin in the test material. Method.   The method according to any one of the preceding claims, wherein the method is performed in combination with other diagnostic methods for colorectal adenoma and / or colorectal carcinoma to increase sensitivity and / or specificity.   Use of transthyretin as a biomarker for the detection of colorectal adenoma and / or colorectal carcinoma in an individual.   20. Use according to claim 19 for the early detection of colorectal adenoma and / or colorectal carcinoma.   21. Use according to claim 19 or 20, wherein the transthyretin is notched, modified and / or mutated.   Use according to any one of claims 19 to 21, wherein the sensitivity and / or specificity is increased in combination with one or more further biomarkers for colorectal adenoma and / or colorectal carcinoma.   23. Use according to claim 22, wherein the at least one further biomarker comprises carcinoembryonic antigen (CEA) and its level in the test material is increased by colorectal adenoma and / or colorectal carcinoma.   23. Use according to claim 22, wherein the at least one further biomarker comprises CA 19-9, whose level in the test material is increased by colorectal adenoma and / or colorectal carcinoma.   23. Use according to claim 22, wherein the at least one further biomarker has a molecular weight of 8960 ± 9 Da and its level in the test material is increased by colorectal adenoma and / or colorectal carcinoma. A test system for detecting colorectal adenoma and / or colorectal carcinoma in an individual comprising:
a) an antibody or receptor that binds to an epitope of transthyretin;
b) a solid support that supports the antibody or receptor;
c) a reagent for detecting the binding of the epitope of the transthyretin to the antibody or receptor;
Including testing system.   27. The test system of claim 26, wherein the test system comprises one or more antibodies or receptors for detecting one or more additional biomarkers for colorectal adenoma and / or colorectal carcinoma. An array comprising detection molecules for detecting colorectal adenoma and / or colorectal carcinoma in an individual,
a) a nucleic acid probe immobilized on a solid support for binding to and detecting transthyretin-encoding mRNA, or b) a solid support for binding to and detecting an epitope of transthyretin An immobilized antibody, or c) a receptor immobilized on a solid support for binding to and detecting an epitope of transthyretin,
Preferably, an array in which different amounts of detection molecules are each immobilized on a solid support to increase the accuracy of quantification.

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